Control of mycotoxin production by chemically inhibiting fungal growth

ABSTRACT

An effective method to control aflatoxin produced by toxic strains of Aspergillus parasiticus fungi is disclosed. An effective amount of Beta-ionone is applied to said fungi to inhibit the growth and sporulation of the fungi and thereby control production of aflatoxin from the fungi without killing the fungi.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 377,509filed May 12, 1982 now abandoned.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to the control of mycotoxins by means of chemicaltreatment.

(2) Description of the Prior Art

Heretofore, it has been known that "turkey X" disease has been caused bya toxin produced by some strains of the fungi Aspergillus parasiticus.This aflatoxin which is produced by strains of A. parasiticus is acutelytoxic as well as carcinogenic. However, much of the research onaflatoxin dealt solely with the detection of aflatoxin and relativelylittle research has been done on the prevention of formation ofaflatoxins.

A review of the control or suppression of fungi producing aflatoxinreveals efforts of fumigating with high level dosages of methyl bromide,ethylene dibromide, propane/propene ethylene oxide, sulfur dioxide, andphosphine and did show some effects of fungicidal activity. Ammoniaproved to be fungicidal but demonstrated a lack of any residual effect.Propionic, acetic, and isobutyric acids also have antifungal activity.However, all the above chemicals have the definite disadvantage oftoxicity to humans and animals, corrosiveness, and lowered nutritionalquality. Therefore, they are not acceptable to either humans or animals.

The problem is magnified because fungal invasion of a crop begins in thefield and either remains or increases during storage. Thus, levels ofaflatoxin usually increase during storage.

SUMMARY OF THE INVENTION

Some strains of Aspergillus parasiticus fungi produce serious amounts ofaflatoxin in agricultural crops during both preharvest and postharvestperiods. This invention consists of a method for inhibiting andcontrolling said aflatoxin which is produced by aflatoxin producingstrains of aspergillus parasiticus fungi and comprises: treating thecrop and associated fungi with an effective amount of Beta-ionone toinhibit and control the growth and sporulation of said fungi withoutfilling the fungi, and thus eliminate the aflatoxin which would beproduced by the fungi is left untreated. Effective amounts ofbeta-ionone have been determined to be about 1 μL to 100 μl.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Aspergillus parasiticus Speare is on deposit with the AgriculturalResearch Culture Collection (NRRL) in Peoria, Illinois, and has beenassigned the following accession number: NRRL 2999. The address of theAgricultural Research Culture Collection (NRRL) is:

A. J. Lyons, Curator, ARS Patent Collection Culture Collection ResearchNRRC, 1815 N. University Street, Peoria, Ill. 61604.

Beta-ionone has a definite effect on the growth and development of A.parasiticus fungi as demonstrated in the following examples:

EXAMPLE 1

Asperguillus parasiticus Speare (NRRL 2999)was maintained on potatodextrose agar (pda) slants and used in these experiments. The A.parasiticus isolate (NRRL 2999)produces aflatoxins.

Direct contact tests with β-ionone were done using petri dishes (15×100mm) containing 20 ml pda and maintained at 26° C. Varying amounts (1-20μl) of β-ionone were pipetted directly onto the pda surface just beforeinoculation. Some of the cultures were placed in plastic bags duringincubation. Gross observations, diameter measurements and microscopicobservations were periodically taken.

Bioassays of volatile effects of β-ionone were also done using pda individed plates. These bioassays were conducted using the followingprocedure: (a) all 4 quadrants were inoculated with stabs of a sporesuspension; (b) different concentrations of β-ionone (1.50 μl) wereplaced only in 1 quadrant; (c) plates were stored in separate plasticbags and incubated at 26° C.; (d) gross observations and diametermeasurements were recorded after 3, 4, or 5 days of incubation; (e)slides were made and microscopic observations recorded.

Shake cultures of A. parasiticus (NRRL 2999) were used to determine theeffects of β-ionone on growth and aflatoxin production. The medium wasprepared by dissolving 50 g Bacto mycological broth w/low pH (Difco), 15g sucrose and 2 g yeast extract in 1,000 ml H₂ O. One hundred ml of themedium were placed in 125 ml Erlenmeyer flasks and autoclaved. When themedium was cool, varying amounts of β-ionone (0.100 μl/flask) werepipetted into the flasks. All experiments were replicated at least 4times. The cultures were inoculated with a spore suspension of A.parasiticus. The flasks were sealed with aluminum foil and rubber bands.The cultures were grown on a rotary shaker and dry weights taken at1,2,3,4,5,6,7, and 10 days.

Aflatoxin was determined from 7-day cultures after extraction of theliquid medium with CHCl₃. Twenty-five ml, of medium was removed fromeach flask and mixed with 25 ml of saturated sodium chloride solution.This mixture was extracted twice with 25 ml CHCl₃. The CHCl₃ layers werecollected and taken to dryness using a rotary evaporator. The residuewas suspended in 1 ml CHCl₃ then diluted with 1 ml hexane. Thesuspension was placed on a silica gel Sep-Pak (Water Assoc.) and clutedwith 5 ml hexane, 5 ml anhydrous ethyl ether, and 3 ml CHCl₃ /CH₃ OH(90:10). The CHCl₃ /CH₃ OH fraction was collected, taken to drynessunder N₂ and reconstituted in HPLC mobile phase. The aflatoxins weredeterminied by HPLC using the method of Thean et al.

RESULTS AND DISCUSSIONS

Direct contact of A. parasiticus with 1,2.5,5,10,20, and 100 μl ofβ-ionone placed on the surface of pda resulted in severely restrictedgrowth and arrested sporulation. The colonies remained light brown-whiteand the growth habit was compact. No sporulation occurred at levels of 5μl or above even after 4 weeks' incubation. Sporulation occurred after1-2 weeks with 1 and 2.5 pl μl of applied β-ionone. These colonies wererestricted and many conidial heads were atypical. Frequently, vesiclesof reduced size were formed, sometimes with irregular sterigmata. Whenmycelial fragments from any treatments (1-100 μl) were transferred tofresh pda, normal growth and sporulation ocurred, therefore no funguswas killed.

The volatile effects of β-ionone on opposite or adjacent quadrants weresomewhat different than the effects of direct contact. One μl ofβ-ionone produced effects only in the quadrant containing β-ionone; thegrowth and sporulation of A. parasiticus was not affected in the otherquadrants. In plates with 5,10, 15,20,25,30,35,40,45 and 50 μl β-iononein 1 quadrant, an increasing effect on growth and sporulation in otherquadrants was noted in plates containing 5-20 μl of β-ionone. Theeffects of increasing from 5 to 20 μl included increasing restriction ofcolony diameters and decreasing levels of sporulation after 7 days.Radial growth in plates receiving 20-50 μl β-ionone was about half thatof the controls. Little sporulation occurred after 7 days in anyquadrant of these plates as long as they remained in unopened bags.

Direct contact with β-ionone at levels of 1-20 μl, resulted in veryrestricted growth, little or no sporulation, and arrested asexualreproductive development. Few, if any, mature conidia were produced. Theprimary thallus consisted of vegetative hyphae and conidiophore initialsthat were atypical or of reduced size. The volatile effects of β-iononewere evidenced by morphological changes, growth inhibition, andsporulation reduction in adjaent and opposite quadrants. Microscopicobservations included:

Reduced size of vesicle and conidiophore diameter, arrested asexualreproduction with many immature conidiophores; increased vegetativegrowth when compared to direct contact; atypical distribution ofsterigmata, similar to direct contact; elongated, irregular sterigmata;atypical branching of conidiophores and abnormal conidiophoreappearance. These effects are concentration-dependent at levels of 1-5μl/plate for direct contact and at levels of 5-20 μl/plate for volatileeffects in divided plates.

The effects of β-ionone on growth (dry wt) and aflatoxin synthesis of A.parasiticus are given in Table 1.

                  TABLE I                                                         ______________________________________                                        Effects of β-ionone on Growth and Aflatoxin B.sub.1                      Accumulation In Shake Liquid Cultures of                                      Aspergillus parasiticus.                                                      β-Ionone added                                                                           Dry wt  Aflatoxin B.sub.1                                     (μl/l)       (g).sup.b                                                                             (ng/ml).sup.b                                         ______________________________________                                         0              1.92    9528                                                   10             1.93    10200                                                  50             1.50    11240                                                 100             1.23    2496                                                  200             1.29    1568                                                  250             1.02    1368                                                  300             0.79     176                                                  400             0.84     280                                                  500             0.71     16                                                   1000            0.74      2                                                   ______________________________________                                         .sup.b Numbers are averages from four flasks per treatment               

The effects on growth were noticeable beginnin at 50 μl/l of medium.Concentrations above 250 μl/l had little further effect on growth. Theprimary effect of β-ionone on growth in shake culture seemed to be onthe rate of growth; however, sporulation of A. parasiticus in shake orsubmerged culture is inhibited and was not measured. Concentrations of100 μl and above of β-ionone/l inhibited aflatoxin accumulations whereas10 and 50 μl/l slightly stimulated aflatoxin production. This shows thatthe ability of the toxigenic strain of A. parasiticus to produceaflatoxin is not necessarily linked to growth; but aflatoxin synthesismay be positively correlated with the asexual reproductive process.

We claim:
 1. A method for inhibiting aflatoxin which is produced byaflatoxin producing strains of Aspergillus parasiticus fungi, saidmethod comprising: treating the fungi with an effective amount ofbeta-ionone to inhibit and control the growth and sporulation of saidfungi, without killing the fungi, thus eliminating the aflatoxinproduced by said fungi.
 2. The method of claim 1 wherein the effectiveamount of beta-ionone is about 1 μl to 100 μl.
 3. The method of claim 2wherein the Aspergillus parasiticus is NRRL 2999.